Updating the Species Diversity of Pestalotioid Fungi: Four New Species of Neopestalotiopsis and Pestalotiopsis

Pestalotioid fungi are associated with a wide variety of plants around the world as pathogens, endophytes, and saprobes. In this study, diseased leaves and branches of plants were collected from Guizhou and Sichuan in China. Here, the fungal isolates were identified based on a phylogenetic analysis of the internal transcribed spacer region (ITS), the translation elongation factor 1-alpha (tef1-α) and the beta-tubulin (tub2) of ribosomal DNA, and the morphological characteristics. Ten Neopestalotiopsis isolates and two Pestalotiopsis isolates were obtained, and these isolates were further confirmed as four novel species (N. acericola, N. cercidicola, N. phoenicis, and P. guiyangensis) and one known species, N. concentrica.


Introduction
Coelomycetous fungi are a diverse group of asexually reproducing fungi that belong to the phylum Ascomycota [1].This group includes a wide variety of fungi that can be found in different environments, including soil, plant surfaces, and even in association with other organisms [1].Coelomycetous fungi are pathogens of a wide variety of plants, with types such as endophytes, saprophytes, and pathogenic fungi [2].Concerning Sporocadaceae, as a member of coelomycetous fungi, a lot of research has been carried out on their taxonomy and pathology based on phylogeny and morphology, and pestalotioid species are some of the most common pathogenic genera [3].Pestalotioid species are the main group of pathogenic fungi that cause leaf spot disease in plants, and they have been found to cause serious ecological problems [4][5][6][7][8][9][10][11][12].
Species of pestalotioid fungi have various ecological behaviors as plant pathogens, endophytes, or saprobes, and are widespread in temperate and tropical regions [13][14][15].Pestalotiopsis was divided from Pestalotia as a distinct genus, on the basis of varying numbers of conidia, by Steyaert in 1949 [16].Subsequently, Nag Raj [17,18] argued that the categorization of many species in Pestalotiopsis, as delineated by Steyaert, is problematic and pointed out that the type of species associated with Pestalotiopsis must be re-examined.In 2014, Maharachchikumbura et al. [15] reclassified Pestalotiopsis into three genera (Neopestalotiopsis, Pestalotiopsis, and Pseudopestalotiopsis).Neopestalotiopsis differs from Pestalotiopsis and Pseudopestalotiopsis based on its multicolored median cells [15].Pseudopestalotiopsis can be distinguished from Pestalotiopsis by comparing the color of the concolorous median cells for those possessing equally pigmented median cells [15].Currently, the three genera are grouped as Pestalotioid fungi [15].

Sample Collection and Isolation
A survey of plants in the Guizhou and Sichuan provinces of China in 2023 resulted in the collection of 98 samples (20 branches and 25 leaves in Sichuan; 28 branches and 25 leaves in Guizhou) with different symptoms, including 12 samples (10 samples in Sicuan and 2 samples in Guizhou) with typical symptoms.The leaves were isolated using the tissue isolation method [31], the surfaces of the leaves were gently wiped clean with distilled water, and the leaf spots were cut into small pieces (0.1 × 0.2 cm) and sterilized (75% ethanol for 30 s, 5% sodium hypochlorite for 45 s, and rinsed with distilled water).After disinfection, the leaves were placed on dry sterile filter paper to absorb the moisture, and then the leaves were placed on potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per liter) and incubated at 25 °C in the dark until spores germinated.Pure cultures were obtained by cutting off hyphal tips of single germinating conidia, transferring them to new PDA plates, and incubating them in the dark at 25 °C.The surface of the blade was sterilized, and the top layer of fruiting bodies on the diseased branches was scraped off.Then, the fruiting bodies were placed on PDA medium and incubated at 25 °C under dark conditions until the spores germinated.Individual germinated conidia were transferred to fresh PDA plates to obtain pure cultures.Herbarium materials were

Sample Collection and Isolation
A survey of plants in the Guizhou and Sichuan provinces of China in 2023 resulted in the collection of 98 samples (20 branches and 25 leaves in Sichuan; 28 branches and 25 leaves in Guizhou) with different symptoms, including 12 samples (10 samples in Sicuan and 2 samples in Guizhou) with typical symptoms.The leaves were isolated using the tissue isolation method [31], the surfaces of the leaves were gently wiped clean with distilled water, and the leaf spots were cut into small pieces (0.1 × 0.2 cm) and sterilized (75% ethanol for 30 s, 5% sodium hypochlorite for 45 s, and rinsed with distilled water).After disinfection, the leaves were placed on dry sterile filter paper to absorb the moisture, and then the leaves were placed on potato dextrose agar (PDA; 200 g potatoes, 20 g dextrose, 20 g agar per liter) and incubated at 25 • C in the dark until spores germinated.Pure cultures were obtained by cutting off hyphal tips of single germinating conidia, transferring them to new PDA plates, and incubating them in the dark at 25 • C. The surface of the blade was sterilized, and the top layer of fruiting bodies on the diseased branches was scraped off.Then, the fruiting bodies were placed on PDA medium and incubated at 25 • C under dark conditions until the spores germinated.Individual germinated conidia were transferred to fresh PDA plates to obtain pure cultures.Herbarium materials were deposited at the Museum of the Beijing Forestry University (BJFC).The 12 cultures obtained in this study were deposited at the China Forestry Culture Collection Centre, Beijing, China (CFCC).

DNA Extraction, PCR Amplification, and Phylogenetic Analysis
Genomic DNA was extracted from mycelium on the PDA using the CTAB method [32].The PCR mixture consisted of 10 µL TopTaq™ Master Mix, 6 µL nucleasefree H 2 O, 1 µL of each primer, and 2 µL DNA.The samples were made up to the final volume of 20 µL.The amplification of partial gene sequences was carried out using three DNA fragments (ITS, the translation elongation factor 1-alpha (tef1-α), and beta-tubulin (tub2)).ITS used primer sets ITS1/ITS4 [33], tef1-α used primer sets EF1-728F/EF1-1567R [34], and tub2 u primer sets T1/Bt-2b [35].The genes and PCR conditions used for the different genera are listed in Table 1.The DNA was sequenced by the SinoGenoMax Company Limited (Beijing, China).The forward and reverse reads were assembled using Seqman v. 7.1.0.MEGA6 was used to manually compare and check the sequences [36].Ambiguous regions were excluded from the alignments.Phylogenetic analyses were analyzed with Maximum Likelihood analysis (ML) and Bayesian Inference (BI) analysis.Maximum Likelihood analysis (ML) was performed in PhyMLv.7.2.8 [37], and MrBayes v. 3.2.0 was used for Bayesian Inference (BI) analysis [38].Maximum Likelihood analysis (ML) was performed using the GTR site substitution model, and branch support was assessed using the bootstrapping (BS) method with 1000 repetitions [39].Bayesian Inference (BI) analysis was performed using the Markov chain Monte Carlo (MCMC) algorithm [39].For Neopestalotiopsis, the GTR+I model, with a certain proportion of invariant sites, was chosen for ITS, and the HKY+G model with gamma distribution rate was chosen for tef1-α and tub2.The GTR+I+G model was chosen for the ITS of Pestalotiopsis, and the HKY+I+G model was chosen for tef1-α and tub2.Two MCMC chains were run for 1,000,000 generations, starting from the random tree, and the first 25% of the trees were discarded every 1000 generations as the posterior probabilities (BPP).The posterior probabilities (BPP) for each analysis were used to evaluate the remaining trees [39].The resulting trees were observed in Figtree v. 1.3.1.

Phylogenetic Analyses
In this study, we combined the analysis of the concatenated DNA sequence datasets of ITS, tef1-α, and tub2 to construct phylogenetic trees for Neopestalotiopsis and Pestalotiopsis.The combined species phylogeny of Neopestalotiopsis isolates consisted of 147 sequences, including outgroups Pseudopestalotiopsis cocos (culture CBS 272.29), Ps. indica (culture CBS 459.78), and Ps.theae (culture MFLUCC12-0055).A total of 2186 characters (556 in ITS, 882 in tef1-α, and 748 in tub2) were included in the phylogenetic analysis.The ML tree with bootstrap values and BI posterior probabilities is shown in Figure 2. The phylogenetic tree inferred from the concatenated alignment resolved the ten Neopestalotiopsis isolates into four well-supported monophyletic evolutionary branches representing three new and one known species of Neopestalotiopsis, respectively (Figure 2).In Pestalotiopsis, the phylogeny of the Pestalotiopsis isolates consisted of 192 sequences, including the outgroup Neopestalotiopsis magna (culture MFLUCC 12-652).A total of 1919 characters (536 in ITS, 572 in tef1-α, and 811 in tub2) were included in the phylogenetic analysis.Similar tree topologies were obtained using ML and BI methods (Figure 3).The two isolates of Pestalotiopsis decomposed into a well-supported monophyletic evolutionary branch representing a new species of Pestalotiopsis (Figure 3).All of the sequences obtained in this study were submitted to GenBank (Table S1).
Pestalotiopsis decomposed into a well-supported monophyletic evolutionary branch representing a new species of Pestalotiopsis (Figure 3).All of the sequences obtained in this study were submitted to GenBank (Table S1).

Taxonomy
Five pestalotioid species were identified and characterized based on a polyphasic approach.There are three new species of Neopestalotiopsis, identified as N. acericola, N. cercidicola, and N. phoenicis, respectively, and one known species (N.concentrica).One Pestalotiopsis species was identified as P. guiyangensis.For all of these identified taxa, species descriptions and illustrations are given below.
Culture characteristics: The colonies on PDA reached up to 60 mm in diameter after seven days at 25 • C. The colonies were filamentous, with an undulate edge to circular, white, with a dense aerial mycelium on the surface, and the fruiting bodies were black.
Typus: CHINA, Sichuan Province, Guangyuan City, Lizhou District, 105 Notes: Two isolates from our collection developed an independent clade in the phylogenetic tree with 100% ML and 1.00 BI value (Figure 2).N. acericola was phylogenetically close to N. iberica (Figure 2), but there were eight nucleotide differences, including seven in tef1-α (427/435, 98.16%) and one in tub2 (375/376, 99.73%) with N. iberica, and differed in conidia width (N.acericola (6.5-8.0 µm); N. iberica ((7.2) 8.2-8.7 (9.8) µm).Moreover, N. acericola had a smaller fourth cell (3.5-5.5 µm) than N. iberica (4.5-6.6 µm) and three median cells of N. acericola with minutely verruculose or smooth surface, three median cells of N. iberica with a smooth surface.Additionally, Neopestalotiopsis acericola was isolated from the branches of Acer palmatum.Neopestalotiopsis iberica was isolated from the leaves and stems of Eucalyptus globulus [28].Culture characteristics: The colonies on PDA reached up to 59 mm in diameter after seven days at 25 °C.The colonies were filamentous to circular, white, with dense aerial mycelium on the surface, and the reverse color was pale yellow.Fruiting bodies were observed after seven days.
Typus: CHINA, Sichuan Province, Guangyuan City, Lizhou District, 105°51′23″ E, 32°25′04″ N, on leaf spots of Cercis chinensis, Y.X.Li and L. Lin, 9 October 2023 (holotype Notes: Phylogenetic analysis combining the DNA sequence datasets of ITS, tef1-α, and tub2 revealed that N. cercidicola formed a separate branch (BI/ML = 1/100) (Figure 2).N. cercidicola was phylogenetically close to N. haikouensis (Figure 2), but there were five bp different in the concatenated alignment (one nucleotide difference in ITS, 484/485, 99.79%; two in tef1-α, 432/434, 99.53%; and two in tub2, 710/712, 99.71%) with N. haikouensis, and differed in host and culture characteristics (N.cercidicola from leaf spots of Cercis chinensis, colonies filamentous to circular, white, with dense aerial mycelium on the surface, and the reverse color was pale yellow; N. haikouensis from leaf spots of Ilex chinensis, colonies edge undulate, white to gray-white, with moderate aerial mycelium on the surface, and the reverse was similar in color) [40].Notes: Phylogenetic analysis combining the DNA sequence datasets of ITS, tef1-α, and tub2 revealed that N. cercidicola formed a separate branch (BI/ML = 1/100) (Figure 2).N. cercidicola was phylogenetically close to N. haikouensis (Figure 2), but there were five bp different in the concatenated alignment (one nucleotide difference in ITS, 484/485, 99.79%; two in tef1-α, 432/434, 99.53%; and two in tub2, 710/712, 99.71%) with N. haikouensis, and differed in host and culture characteristics (N.cercidicola from leaf spots of Cercis chinensis, colonies filamentous to circular, white, with dense aerial mycelium on the surface, and the reverse color was pale yellow; N. haikouensis from leaf spots of Ilex chinensis, colonies edge undulate, white to gray-white, with moderate aerial mycelium on the surface, and the reverse was similar in color) [40].Notes: N. concentrica was originally described from spines of Rosa rugosa in Henan Province, China [41].In this study, two isolates (CFCC 70629 and CFCC 70619) clustered together with N. concentrica (CFCC 55163, CFCC 55162, and ROC 135) with 67% ML and 0.91% BI value (Figure 2).Therefore, two isolates were identified as N. concentrica (Figure 6) as a new host and novel geographic record for China.Notes: N. concentrica was originally described from spines of Rosa rugosa in Henan Province, China [41].In this study, two isolates (CFCC 70629 and CFCC 70619) clustered together with N. concentrica (CFCC 55163, CFCC 55162, and ROC 135) with 67% ML and 0.91% BI value (Figure 2).Therefore, two isolates were identified as N. concentrica (Figure 6) as a new host and novel geographic record for China.Culture characteristics: The colonies on PDA reached up to 55 mm in diameter after seven days at 25 °C.The colonies were filamentous to circular, with dense aerial mycelium on surface, and white from above and reverse.Fruiting bodies were observed after ten days.

Discussion
In this study, we investigated pestalotioid fungi of plants in Sichuan and Guizhou provinces in China.We included ten sequenced isolates of Neopestalotiopsis and two

Discussion
In this study, we investigated pestalotioid fungi of plants in Sichuan and Guizhou provinces in China.We included ten sequenced isolates of Neopestalotiopsis and two sequenced isolates of Pestalotiopsis to provide a backbone tree for the genera Neopestalotiopsis and Pestalotiopsis.In this study, four new species (N.acericola, N. cercidicola, N. phoenicis, and P. guiyangensis) and one known species (N.concentrica) were identified on the basis of morphological and phylogenetic analyses.
Pestalotioid fungi are important pathogens of plant diseases, and it has been shown that pestalotioid fungi have no host specificity [42].For example, Pestalotiopsis chamaeropis infests plants in China such as Quercus acutissima, Rosa chinensis, and Camellia sinensis [42]; while Neopestalotiopsis rhapidis was found to infest plants in China such as Rhapis excelsa and Podocarpus macrophyllus [20].In this study, the coelomycetous fungi were found to infest Rhapis excelsa in addition to infesting Rosa rugosa in China [41].The new species, Pestalotiopsis guiyangensis, in this study was found to infest different hosts (Eriobotrya japonica and Rohdea japonica).This indicates that there may be a high undescribed diversity of fungi and host diversity in plants in China [41].
The present study provides a further extension of the backbone tree of Neopestalotiopsis and Pestalotiopsis, and future work requires further investigation in order to establish a more stable backbone tree of Neopestalotiopsis and Pestalotiopsis.More sampling is needed in the future to determine the spread and epidemiology of pestalotioid fungi in Guizhou and Sichuan, China.Therefore, future work needs to be carried out on pathogenicity testing and disease control methods, as this can help us to prevent diseases caused by pestalotioid fungi.
Sequence data are essential to resolve these three genera, as many pestalotioid species have overlapping morphological traits [14].A gene-by-gene assessment of phylogenetic resolution can yield higher levels of protein genes than those of ribosomal regions [43].Hu et al. [44] suggested that at least two gene combinations (ITS and tub2) should be used to resolve the phylogeny of species of Neopestalotiopsis and Pestalotiopsis.Maharachchikumbura et al. [14] tested ten gene regions to resolve the bound species in Neopestalotiopsis and Pestalotiopsis, and finally screened the three most applicable regions (ITS, tef1-α, and tub2) [14].Liu F et al. [45] investigated a genome-wide phylogenetic tree based on Colletotrichum, which could better define the Colletotrichum species complex boundaries.Considering the importance of pestalotioid fungi, genome sequencing of pestalotioid species is recommended.This will not only pave the way for comprehensively resolving the tree of life of Neopestalotiopsis and Pestalotiopsis, but also provide important data for revealing their evolution and adaptive mechanisms and improve our understanding of the genetic basis of various biological features and metabolic potential of these fungi.

Figure 1 .
Figure 1.Diseased plants in Sichuan and Guizhou: (A) Symptoms of Rhapis excelsa in Sichuan; (B) Leaf spots of Phoenix canariensis in Sichuan; (C) Branch dieback of Acer palmatum in Sichuan; (D) Pathogenic fungi on Cercis chinensis leaves in Guizhou; (E) Leaf spots of Eriobotrya japonica in Guizhou.

Figure 1 .
Figure 1.Diseased plants in Sichuan and Guizhou: (A) Symptoms of Rhapis excelsa in Sichuan; (B) Leaf spots of Phoenix canariensis in Sichuan; (C) Branch dieback of Acer palmatum in Sichuan; (D) Pathogenic fungi on Cercis chinensis leaves in Guizhou; (E) Leaf spots of Eriobotrya japonica in Guizhou.

Figure 2 .
Figure 2. Phylograms were generated by maximum likelihood (ML) based on combined ITS, tef1-α, and tub2 sequence data of Neopetalotiopsis isolates.The tree was rooted by Ps. cocos (CBS 272.29),Ps. indica (CBS 459.78), and Ps.theae (MFLUCC12-0055).Scale bars indicate 0.02 nucleotide changes per locus.ML bootstrap support values above 70% are shown near nodes.Thickened branches represent posterior probabilities above 0.95 from BI. Isolates from this study are marked in blue in the trees.Ex-type strains are in bold and labeled T.

Figure 3 .
Figure 3. Phylograms were generated by maximum likelihood (ML) based on combined ITS, tef1-α, and tub2 sequence data of Pestalotiopsis isolates.The tree was rooted by N. magna (MFLUCC 12-652).Scale bars indicate 0.05 nucleotide changes per locus.ML bootstrap support values above 70% are shown near nodes.Thickened branches represent posterior probabilities above 0.95 from BI. Isolates from this study are marked in blue in the trees.Ex-type strains are in bold and labeled T.

Table 1 .
Gene fragments and PCR thermal cycle program used in this study.